Dosage and administration of non-fucosylated anti-cd40 antibody

ABSTRACT

This invention relates methods of using a non-fucosylated anti-CD40 antibody for treatment of cancer and chronic infectious diseases.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 17/734,541, filed on May 2, 2022, which is a divisional applicationof U.S. application Ser. No. 17/535,076, filed on Nov. 24, 2021, whichis a divisional of U.S. application Ser. No. 15/522,614, filed on Apr.27, 2017, which is a U.S. national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2015/058108, filed Oct. 29, 2015,which claims the benefit of U.S. Provisional Patent Application No.62/072,031, filed Oct. 29, 2014 and U.S. Provisional Patent ApplicationNo. 62/134,955, filed on Mar. 18, 2015, each of which are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

This application includes an electronic sequence listing in a file named49223-0024004_SL_ST26.xml created on Nov. 8, 2022 and containing 4197bytes which is hereby incorporated by reference.

FIELD OF THE INVENTION

This disclosure relates methods of using a non-fucosylated anti-CD40antibody for treatment of cancer and chronic infectious diseases.

BACKGROUND OF THE INVENTION

CD40 is a member of the tumor necrosis factor (TNF) receptorsuperfamily. It is a single chain type I transmembrane protein with anapparent MW of 50 kDa. CD40 is expressed by some cancer cells, e.g.,lymphoma cells and several types of solid tumor cells. CD40 alsofunctions to activate the immune system by facilitatingcontact-dependent reciprocal interaction between antigen-presentingcells and T cells. Although a number of anti-CD40 antibodies have beentested in clinical trials, to date none have exhibited sufficientactivity. The present disclosure solves this and other problems.

BRIEF SUMMARY OF THE INVENTION

This disclosure provides a method of treating cancer, by administeringan anti-CD40 antibody to a patient in need of such treatment. Theanti-CD40 antibody comprises the heavy chain variable region of SEQ IDNO:1 and the light chain variable region of SEQ ID NO:2, and a humanconstant region. The constant region has an N-glycoside-linked sugarchain at residue N297 according to the EU index as set forth in Kabatand less than 5% of the N-glycoside-linked sugar chains include a fucoseresidue, i.e., a fucose bound to the reducing terminal of the sugarchain via an α1,6 bond to N-acetylglucosamine (“GlcNAc”). Administrationof the anti-CD40 antibody is at a dose level between 0.1-300 μg/kg (μgantibody per kilogram patient body weight). In one embodiment, theanti-CD40 antibody dose level is between 0.6-150 μg/kg. In anotherembodiment, the anti-CD40 antibody dose level is between 1.0-100 μg/kg.In another embodiment, the anti-CD40 antibody dose level is between 5-25μg/kg. In another embodiment, the anti-CD40 antibody dose level isbetween 8-12 μg/kg. In another embodiment, the anti-CD40 antibody doselevel is about 10 μg/kg. In another embodiment, the anti-CD40 antibodythe dose level is 10 μg/kg.

In another aspect, this disclosure provides a method of treating cancer,by administering an anti-CD40 antibody to a patient in need of suchtreatment. The anti-CD40 antibody comprises the heavy chain variableregion of SEQ ID NO:1 and the light chain variable region of SEQ IDNO:2, and a human constant region. The constant region has anN-glycoside-linked sugar chain at residue N297 according to the EU indexas set forth in Kabat and less than 5% of the N-glycoside-linked sugarchains include a fucose residue, i.e., a fucose bound to the reducingterminal of the sugar chain via an α1,6 bond to N-acetylglucosamine(“GlcNAc”). Administration of the anti-CD40 antibody is at a dose levelbetween 0.1-2000 μg/kg (μg antibody per kilogram patient body weight).In one embodiment, the dose level is between 10-1000 μg/kg. In anotherembodiment, the dose level is between 50-800 μg/kg. In a furtherembodiment, the dose level is between 75-600 μg/kg. In anotherembodiment, the dose level is between 100-500 μg/kg. in furtherembodiments, the dose level is a range selected from the following:100-300 μg/kg, 300-500 μg/kg, 500-700 μg/kg, 700-900 μg/kg, and 900-1100μg/kg. In other embodiments, the dose level is a range selected from thefollowing: 100-150 μg/kg, 150-200 μg/kg, 200-250 μg/kg, 250-300 μg/kg,300-350 μg/kg, 350-400 μg/kg, 400-450 μg/kg, 450-500 μg/kg, 500-550μg/kg, 550-600 μg/kg, 600-650 μg/kg, 650-700 μg/kg, 700-750 μg/kg,750-800 μg/kg, 800-850 μg/kg, 850-900 μg/kg, 900-950 μg/kg, 950-1000μg/kg, 1000-1050 μg/kg, and 1050-1100 μg/kg. In further embodiments, thedose level is selected from the following: about 60 μg/kg, about 100μg/kg, about 150 μg/kg, about 200 μg/kg, about 250 μg/kg, about 300μg/kg, about 350 μg/kg, about 400 μg/kg, about 450 μg/kg, about 500μg/kg, about 550 μg/kg, about 600 μg/kg, about 650 μg/kg, about 700μg/kg, about 750 μg/kg, about 800 μg/kg, about 850 μg/kg, about 900μg/kg, about 950 μg/kg, about 1000-1050 μg/kg, about 1050 μg/kg, and1110 μg/kg.

In one embodiment, the anti-CD40 antibody is administered every threeweeks. In another embodiment the anti-CD40 antibody is administeredevery six weeks. In another embodiment the anti-CD40 antibody isadministered every ten weeks. In another embodiment the anti-CD40antibody is administered every twelve weeks. In another embodiment theanti-CD40 antibody is administered every fifteen weeks. In anotherembodiment the anti-CD40 antibody is administered every eighteen weeks.

In another embodiment, the patient has a CD40 positive cancer. Inanother embodiment, the patient has a CD40 negative cancer. In a furtherembodiment, the patient has a cancer that is a solid tumor. In yetanother embodiment, the patient has a cancer that is a blood cancer. Inanother embodiment, the cancer is a melanoma, a breast cancer, includingmetastatic breast cancer, a lung cancer, including a non-small cell lungcancer, or pancreatic cancer.

In a further aspect, this disclosure provides methods of treating cancerby administering to the patient a combination of the anti-CD40 antibodyand an antibody that blocks an immune checkpoint. One example of anantibody that blocks an immune checkpoint is an anti-cytotoxicT-lymphocyte-associated protein 4 (CTLA4) antibody. Examples ofanti-CTLA4 antibodies include, e.g., ipilimumab or tremelimumab. Anotherexample of an antibody that blocks an immune checkpoint is ananti-programmed cell death protein 1 (PD1) antibody. Examples ofanti-PD1 antibodies include, e.g., nivolumab, pidilizumab, orpembrolizumab. A further example of an antibody that blocks an immunecheckpoint is an anti-programmed death-ligand (PD-L1) antibody. Examplesof anti-PD-L1 antibodies include, e.g., MEDI4736 and MPDL3280A.

In another embodiment, the patient has a CD40 positive cancer and istreated with a combination of the anti-CD40 antibody and an antibodythat blocks an immune checkpoint, e.g., an anti-CTLA4 antibody, ananti-PD1 antibody, or an anti-PD-L1 antibody. In another embodiment, thepatient has a CD40 negative cancer and is treated with a combination ofthe anti-CD40 antibody and an antibody that blocks an immune checkpoint,e.g., an anti-CTLA4 antibody, an anti-PD1 antibody, or an anti-PD-L1antibody. In a further embodiment, the patient has a cancer that is asolid tumor and is treated with a combination of the anti-CD40 antibodyand an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4antibody, an anti-PD1 antibody, or an anti-PD-L1 antibody. In yetanother embodiment, the patient has a cancer that is a blood cancer andis treated with a combination of the anti-CD40 antibody and an antibodythat blocks an immune checkpoint, e.g., an anti-CTLA4 antibody, ananti-PD1 antibody, or an anti-PD-L1 antibody. In another embodiment, thecancer is a melanoma, a breast cancer, including metastatic breastcancer, a lung cancer, including a non-small cell lung cancer, orpancreatic cancer, and is treated with a combination of the anti-CD40antibody and an antibody that blocks an immune checkpoint, e.g., ananti-CTLA4 antibody, an anti-PD1 antibody, or an anti-PD-L1 antibody.

Definitions

A “polypeptide” or “polypeptide chain” is a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically. Polypeptides of less than about 10 amino acid residuesare commonly referred to as “peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “antibody” is used herein to denote immunoglobulin proteinsproduced by the body in response to the presence of an antigen and thatbind to the antigen, as well as antigen-binding fragments and engineeredvariants thereof. Hence, the term “antibody” includes, for example,intact monoclonal antibodies comprising full-length immunoglobulin heavyand light chains (e.g., antibodies produced using hybridoma technology)and antigen-binding antibody fragments, such as F(ab′)₂ and Fabfragments. Genetically engineered intact antibodies and fragments, suchas chimeric antibodies, humanized antibodies, single-chain Fv fragments,single-chain antibodies, diabodies, minibodies, linear antibodies,multivalent or multispecific (e.g., bispecific) hybrid antibodies, andthe like are also included. Thus, the term “antibody” is usedexpansively to include any protein that comprises an antigen-bindingsite of an antibody and is capable of specifically binding to itsantigen.

An “antigen-binding site of an antibody” is that portion of an antibodythat is sufficient to bind to its antigen. The minimum such region istypically a variable domain or a genetically engineered variant thereof.Single-domain binding sites can be generated from camelid antibodies(see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyenet al., EMBO J. 19:921-930, 2000) or from VH domains of other species toproduce single-domain antibodies (“dAbs”; see Ward et al., Nature341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In certainvariations, an antigen-binding site is a polypeptide region having only2 complementarity determining regions (CDRs) of a naturally ornon-naturally (e.g., mutagenized) occurring heavy chain variable domainor light chain variable domain, or combination thereof (see, e.g., Pessiet al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol.25:921-929, 2007). More commonly, an antigen-binding site of an antibodycomprises both a heavy chain variable (VH) domain and a light chainvariable (VL) domain that bind to a common epitope. Within the contextof the present invention, an antibody may include one or more componentsin addition to an antigen-binding site, such as, for example, a secondantigen-binding site of an antibody (which may bind to the same or adifferent epitope or to the same or a different antigen), a peptidelinker, an immunoglobulin constant region, an immunoglobulin hinge, anamphipathic helix (see Pack and Pluckthun, Biochem. 31:1579-1584, 1992),a non-peptide linker, an oligonucleotide (see Chaudri et al., FEBSLetters 450:23-26, 1999), a cytostatic or cytotoxic drug, and the like,and may be a monomeric or multimeric protein. Examples of moleculescomprising an antigen-binding site of an antibody are known in the artand include, for example, Fv, single-chain Fv (scFv), Fab, Fab′,F(ab′)₂, F(ab)_(c), diabodies, dAbs, minibodies, nanobodies, Fab-scFvfusions, bispecific (scFv)₄-IgG, and bispecific (scFv)₂-Fab. (See, e.g.,Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., MolecularImmunology 33:1301-1312, 1996; Carter and Merchant, Curr. Opin.Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367,2000; and Lu et al., J. Immunol. Methods 267:213-226, 2002.)

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingene(s). One form of immunoglobulin constitutes the basic structuralunit of native (i.e., natural) antibodies in vertebrates. This form is atetramer and consists of two identical pairs of immunoglobulin chains,each pair having one light chain and one heavy chain. In each pair, thelight and heavy chain variable regions (VL and VH) are togetherprimarily responsible for binding to an antigen, and the constantregions are primarily responsible for the antibody effector functions.Five classes of immunoglobulin protein (IgG, IgA, IgM, IgD, and IgE)have been identified in higher vertebrates. IgG comprises the majorclass; it normally exists as the second most abundant protein found inplasma. In humans, IgG consists of four subclasses, designated IgG1,IgG2, IgG3, and IgG4. The heavy chain constant regions of the IgG classare identified with the Greek symbol γ. For example, immunoglobulins ofthe IgG1 subclass contain a γ1 heavy chain constant region. Eachimmunoglobulin heavy chain possesses a constant region that consists ofconstant region protein domains (CH1, hinge, CH2, and CH3; IgG3 alsocontains a CH4 domain) that are essentially invariant for a givensubclass in a species. DNA sequences encoding human and non-humanimmunoglobulin chains are known in the art. (See, e.g., Ellison et al.,DNA 1:11-18, 1981; Ellison et al., Nucleic Acids Res. 10:4071-4079,1982; Kenten et al., Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Senoet al., Nuc. Acids Res. 11:719-726, 1983; Riechmann et al., Nature332:323-327, 1988; Amster et al., Nuc. Acids Res. 8:2055-2065, 1980;Rusconi and Kohler, Nature 314:330-334, 1985; Boss et al., Nuc. AcidsRes. 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982; vander Loo et al., Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol.Evol. 22:195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982; Breineret al., Gene 18:165-174, 1982; Kondo et al., Eur. J. Immunol.23:245-249, 1993; and GenBank Accession No. J00228.) For a review ofimmunoglobulin structure and function, see Putnam, The Plasma Proteins,Vol V, Academic Press, Inc., 49-140, 1987; and Padlan, Mol. Immunol.31:169-217, 1994. The term “immunoglobulin” is used herein for itscommon meaning, denoting an intact antibody, its component chains, orfragments of chains, depending on the context.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the amino-terminus(encoding about 110 amino acids) and a by a kappa or lambda constantregion gene at the carboxyl-terminus. Full-length immunoglobulin “heavychains” (about 50 Kd or 446 amino acids) are encoded by a variableregion gene (encoding about 116 amino acids) and a gamma, mu, alpha,delta, or epsilon constant region gene (encoding about 330 amino acids),the latter defining the antibody's isotype as IgG, IgM, IgA, IgD, orIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. (See generally Fundamental Immunology (Paul, ed., RavenPress, N.Y., 2nd ed. 1989), Ch. 7).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three hypervariable regions, also called“complementarity determining regions” or “CDRs.” The framework regionsserve to align the CDRs for specific binding to an epitope of anantigen. Thus, the term “hypervariable region” or “CDR” refers to theamino acid residues of an antibody that are primarily responsible forantigen binding. From amino-terminus to carboxyl-terminus, both VL andVH domains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989. Kabat alsoprovides a widely used numbering convention (Kabat numbering) in whichcorresponding residues between different heavy chains or betweendifferent light chains are assigned the same number. CDRs 1, 2, and 3 ofa VL domain are also referred to herein, respectively, as CDR-L1,CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred toherein, respectively, as CDR-H1, CDR-H2, and CDR-H3.

Unless the context dictates otherwise, the term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

The term “chimeric antibody” refers to an antibody having variabledomains derived from a first species and constant regions derived from asecond species. Chimeric immunoglobulins or antibodies can beconstructed, for example by genetic engineering, from immunoglobulingene segments belonging to different species. The term “humanizedantibody,” as defined infra, is not intended to encompass chimericantibodies. Although humanized antibodies are chimeric in theirconstruction (i.e., comprise regions from more than one species ofprotein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not foundin chimeric immunoglobulins or antibodies, as defined herein.

The term “humanized VH domain” or “humanized VL domain” refers to animmunoglobulin VH or VL domain comprising some or all CDRs entirely orsubstantially from a non-human donor immunoglobulin (e.g., a mouse orrat) and variable region framework sequences entirely or substantiallyfrom human immunoglobulin sequences. The non-human immunoglobulinproviding the CDRs is called the “donor” and the human immunoglobulinproviding the framework is called the “acceptor.” In some instances,humanized antibodies may retain non-human residues within the humanvariable domain framework regions to enhance proper bindingcharacteristics (e.g., mutations in the frameworks may be required topreserve binding affinity when an antibody is humanized).

A “humanized antibody” is an antibody comprising one or both of ahumanized VH domain and a humanized VL domain. Immunoglobulin constantregion(s) need not be present, but if they are, they are entirely orsubstantially from human immunoglobulin constant regions.

Specific binding of an antibody to its target antigen means an affinityof at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding isdetectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not, however, necessarily imply that a monoclonal antibody bindsone and only one target.

With regard to proteins as described herein, reference to amino acidresidues corresponding to those specified by SEQ ID NO includespost-translational modifications of such residues.

The term “diluent” as used herein refers to a solution suitable foraltering or achieving an exemplary or appropriate concentration orconcentrations as described herein.

The term “container” refers to something into which an object or liquidcan be placed or contained, e.g., for storage (for example, a holder,receptacle, vessel, or the like).

The term “administration route” includes art-recognized administrationroutes for delivering a therapeutic protein such as, for example,parenterally, intravenously, intramuscularly, or subcutaneously. Foradministration of an antibody for the treatment of cancer,administration into the systemic circulation by intravenous orsubcutaneous administration may be desired. For treatment of a cancercharacterized by a solid tumor, administration can also be localizeddirectly into the tumor, if so desired.

The term “treatment” refers to the administration of a therapeutic agentto a patient, who has a disease with the purpose to cure, heal,alleviate, delay, relieve, alter, remedy, ameliorate, improve or affectthe disease.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The term “effective amount,” “effective dose,” or “effective dosage”refers to an amount that is sufficient to achieve or at least partiallyachieve the desired effect, e.g., sufficient to inhibit the occurrenceor ameliorate one or more symptoms of a disease or disorder. Aneffective amount of a pharmaceutical composition is administered in an“effective regime.” The term “effective regime” refers to a combinationof amount of the composition being administered and dosage frequencyadequate to accomplish prophylactic or therapeutic treatment of thedisease or disorder.

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20μg/Kg” encompasses a range of 18-22 μg/Kg. As used herein, about alsoincludes the exact amount. Hence “about 20 μg/Kg” means “about 20 μg/Kg”and also “20 μg/Kg.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the binding of SEA-CD40 (solid line) and dacetuzumab(dashed line) for the human CD40 protein present on the surface ofPBMCs.

FIGS. 2A and 2B provides the binding affinities of SEA-CD40 (open andclosed squares) and dacetuzumab (open and closed circles) for the humanFcγIIIa receptor variants. FIG. 2A provides a graphical representationand FIG. 2B provides K_(D) values. SEA-CD40 values are shown in the leftcolumn; decetuzumab values are shown in the right column.

FIG. 3 provides a dose relationship and time course of B-cell depletionfrom human peripheral blood mononuclear cells (PBMCs) as a result oftreatment with SEA-CD40.

FIGS. 4A and 4B demonstrate representative cytokine production by humanwhole blood after twenty-four hours of treatment with SEA-CD40 or anisotype control (SEA-h00). Antibodies were adminstered in units ofμg/ml. FIG. 4A shows production of tumor necrosis factor-α and FIG. 4Bshows production of MIP-1β.

FIGS. 5A and 5B demonstrate representative cytokine production by humanPBMCs after twenty-four hours of treatment with SEA-CD40 or an isotypecontrol (SEA-h00). Antibodies were adminstered in units of μg/ml. FIG.5A shows production of tumor necrosis factor-α (TNF-α) and FIG. 5B showsproduction of MIP-1β.

FIG. 6 provides a time course of B-cell depletion from human PBMCs as aresult of treatment with SEA-CD40 (closed squares); dacetuzumab (greycircles); or SEA-CD40 F(ab′)₂ (grey squares).

FIG. 7 provides interferon-γ (IFNγ) production by PBMCs as a result oftreatment with SEA-CD40 (closed squares); dacetuzumab (grey circles); orSEA-CD40 F(ab′)₂ (grey squares).

FIG. 8 demonstrates induction of HLA-DR/DQ/DP as a marker for antigenpresenting cell maturation by PBMCs as a result of treatment withSEA-CD40 (closed squares); dacetuzumab (grey circles), or SEA-CD40F(ab′)₂ (grey squares).

FIG. 9 provides concentration vs. normalized response curves for immuneactivation markers in PBMCs treated with varying concentration ofSEA-CD40.

FIGS. 10A and 10B compare the immune response to the M1 flu peptide byPBMCs incubated with SEA-CD40 or dacetuzumab. FIG. 10A shows levelspercentages of antigen specific T-cells; FIG. 10B shows levels of IFN-γproduction.

FIG. 11 demonstrates enhancement of the immune response to the M1 flupeptide by PBMCs incubated with a combination of SEA-CD40 and either ananti-CTLA-4 antibody or an anti-PD-1 antibody. IFNγ levels are shown inFIG. 11 .

FIG. 12 demonstrates enhancement of the immune response to the M1 flupeptide by PBMCs incubated with a combination of SEA-CD40 and either ananti-CTLA-4 antibody or an anti-PD-1 antibody. Levels of antigenspecific T cells are shown in FIG. 12 .

FIG. 13 provides the immune response (IFNγ production) of PBMCs fromdonors with cancer to common tumor antigen peptides(MAGEA1/MAGE3/NY-ESO). PBMC's were incubated in the presence or absenceof increasing concentrations of SEA-CD40 or SGN-40 for 5 days.

FIG. 14 provides the immune response (IFNγ production) of PBMCs fromdonors with cancer to common tumor antigen peptides(MAGEA1/MAGE3/NY-ESO). PBMC's were incubated in the presence or absenceof increasing concentrations of SEA-CD40 and/or a constant concentrationof an anti-CTLA4 or anti-PD1 blocking antibody.

FIGS. 15A and 15B demonstrate the binding of fucosylated andnon-fucosylated anti-mouse CD40 antibodies to murine Fcγ receptors. Fcγreceptor were either FcγRI (FIG. 15A) or FcγRIV (FIG. 15B).

FIG. 16 demonstrates in vivo activity of fucosylated and non-fucosylatedanti-CD40 antibody surrogates in the mouse B16 melanoma model.

FIG. 17 demonstrates B-cell activation activity of SEA-CD40, antibody21.4.1, and CD40 hexameric ligand. Experiments were performed usingpurified B-cell cultures.

FIG. 18 demonstrates B-cell activation activity of SEA-CD40, antibody21.4.1, and CD40 hexameric ligand. Experiments were performed using PBMCcultures.

FIG. 19 demonstrates monocyte/macrophage activation activity ofSEA-CD40, antibody 21.4.1, dacetuzumab and an SEA-isotype control.

FIG. 20 demonstrates induction of interferon-γ (IFN-γ) levels bySEA-CD40, antibody 21.4.1, dacetuzumab or an SEA-isotype control.

FIG. 21 demonstrates induction of interleukin 10 (IL10) levels bySEA-CD40, antibody 21.4.1, dacetuzumab or an SEA-isotype control.

FIG. 22 demonstrates induction of interferon-γ (IFN-γ) levels bySEA-CD40, antibody 21.4.1, or dacetuzumab. Incubation was done in thepresence of flu peptide.

FIG. 23 demonstrates induction a flu-antigen specific T-cell response bySEA-CD40, antibody 21.4.1, or dacetuzumab.

FIG. 24 demonstrates changes in IL10 levels following incubation ofPBMCs with flu peptide and SEA-CD40, antibody 21.4.1, or dacetuzumab.

DETAILED DESCRIPTION

This disclosure provides description of the activity of anon-fucosylated anti-CD40 antibody, SEA-CD40. SEA-CD40 is an agonisticantibody and has enhanced binding to Fcγ receptors III and, surprisinglyexhibits enhanced activation of the CD40 signaling pathway. Because ofits enhanced activation of the CD40 pathway SEA-CD40 is a potentactivator of the immune system and can be used to treat cancer or totreat infectious diseases, particularly chronic viral diseases, such ashepatitis C, human immunodeficiency virus, Epstein-Barr virus,cytomegalovirus, John Cunningham virus, and human papilloma virus. Otherinfectious diseases, include, e.g., tuberculosis. The enhancedactivation of the immune system allows SEA-CD40 to be dosed at lowlevels, as compared to a fucosylated parent antibody.

CD40 Description and Function.

CD40 is a member of the tumor necrosis factor (TNF) receptorsuperfamily. It is a single chain type I transmembrane protein with anapparent MW of 50 kDa. Its mature polypeptide core consists of 237 aminoacids, of which 173 amino acids comprise an extracellular domain (ECD)organized into 4 cysteine-rich repeats that are characteristic of TNFreceptor family members. Two potential N-linked glycosylation sites arepresent in the membrane proximal region of the ECD, while potentialO-linked glycosylation sites are absent. A 22 amino acid transmembranedomain connects the ECD with the 42 amino acid cytoplasmic tail of CD40.Sequence motifs involved in CD40-mediated signal transduction have beenidentified in the CD40 cytoplasmic tail. These motifs interact withcytoplasmic factors called TNF-R-associated factors (TRAFs) to triggermultiple downstream events including activation of MAP kinases and NFκB,which in turn modulate the transcriptional activities of a variety ofinflammation-, survival-, and growth-related genes. See, e.g., vanKooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al.,Immunol. Rev. 229:152-172 (2009).

Within the hematopoietic system, CD40 can be found on B cells atmultiple stages of differentiation, monocytes, macrophages, platelets,follicular dendritic cells, dendritic cells (DC), eosinophils, andactivated T cells. In normal non-hematopoietic tissues, CD40 has beendetected on renal epithelial cells, keratinocytes, fibroblasts ofsynovial membrane and dermal origins, and activated endothelium. Asoluble version of CD40 is released from CD40-expressing cells, possiblythrough differential splicing of the primary transcript or limitedproteolysis by the metalloproteinase TNFα converting enzyme. Shed CD40can potentially modify immune responses by interfering with theCD40/CD40L interaction. See, e.g., van Kooten and Banchereau, J. Leukoc.Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009).

The endogenous ligand for CD40 (CD40L) is a type II membraneglycoprotein of 39 kDa also known as CD154. CD40L is a member of the TNFsuperfamily and is expressed as a trimer on the cell surface. CD40L istransiently expressed on activated CD4+, CD8+, and γδ T cells. CD40L isalso detected at variable levels on purified monocytes, activated Bcells, epithelial and vascular endothelial cells, smooth muscle cells,and DCs, but the functional relevance of CD40L expression on these celltypes has not been clearly defined (van Kooten 2000; Elgueta 2009).However, expression of CD40L on activated platelets has been implicatedin the pathogenesis of thrombotic diseases. See, e.g., Ferroni et al.,Curr. Med. Chem. 14:2170-2180 (2007).

The best-characterized function of the CD40/CD40L interaction is itsrole in contact-dependent reciprocal interaction betweenantigen-presenting cells and T cells. See, e.g., van Kooten andBanchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol.Rev. 229:152-172 (2009). Binding of CD40L on activated T cells to CD40on antigen-activated B cells not only drives rapid B cell expansion, butalso provides an essential signal for B cells to differentiate intoeither memory B cells or plasma cells. CD40 signaling is responsible forthe formation of germinal centers in which B cells undergo affinitymaturation and isotype switching to acquire the ability to produce highaffinity antibodies of the IgG, IgA, and IgE isotypes. See, e.g., Kehry,J. Immunol. 156:2345-2348 (1996). Thus, individuals with mutations inthe CD40L locus that prevent functional CD40/CD40L interaction sufferfrom the primary immunodeficiency X-linked hyper-IgM syndrome that ischaracterized by over-representation of circulating IgM and theinability to produce IgG, IgA, and IgE. These patients demonstratesuppressed secondary humoral immune responses, increased susceptibilityto recurrent pyrogenic infections, and a higher frequency of carcinomasand lymphomas. Gene knockout experiments in mice to inactivate eitherCD40 or CD40L locus reproduce the major defects seen in X-linkedhyper-IgM patients. These KO mice also show impaired antigen-specific Tcell priming, suggesting that the CD40L/CD40 interaction is also acritical factor for mounting cell-mediated immune responses. See, e.g.,Elgueta et al., Immunol. Rev. 229:152-172 (2009).

The immune-stimulatory effects of CD40 ligation by CD40L or anti-CD40 invivo have correlated with immune responses against syngeneic tumors.See, e.g., French et al., Nat. Med. 5:548-553 (1999). A deficient immuneresponse against tumor cells may result from a combination of factorssuch as expression of immune checkpoint molecules, such as PD-1 orCTLA-4, decreased expression of MHC antigens, poor expression oftumor-associated antigens, appropriate adhesion, or co-stimulatorymolecules, and the production of immunosuppressive proteins like TGFβ bythe tumor cells. CD40 ligation on antigen presenting and transformedcells results in up-regulation of adhesion proteins (e.g., CD54),co-stimulatory molecules (e.g., CD86) and MHC antigens, as well asinflammatory cytokine secretion, thereby potentially inducing and/orenhancing the antitumor immune response, as well as the immunogenicityof the tumor cells. See, e.g., Gajewski et al., Nat. Immunol.14:1014-1022 (2013).

A primary consequence of CD40 cross-linking is DC activation (oftentermed licensing) and potentiation of myeloid and B cells ability toprocess and present tumor-associated antigens to T cells. Besides havinga direct ability to activate the innate immune response, a uniqueconsequence of CD40 signaling is APC presentation of tumor-derivedantigens to CD8+ cytotoxic T cell (CTL) precursors in a process known as‘cross-priming’. This CD40-dependent activation and differentiation ofCTL precursors by mature DCs into tumor-specific effectors CTLs mayenhance cell-mediated immune responses against tumor cells. See, e.g.,Kurts et al., Nat. Rev. Immunol. 10:403-414 (2010).

Agonistic CD40 mAbs including dacetuzumab, the SEA-CD40 parent molecule,have shown encouraging clinical activity in single-agent and combinationchemotherapy settings. Dacetuzumab demonstrated some clinical activityin a phase 1 study in NHL and a phase 2 study in diffuse large B-celllymphoma (DLBCL). See, e.g., Advani et al., J. Clin. Oncol. 27:4371-4377(2009) and De Vos et al., J. Hematol. Oncol. 7:1-9 (2014). AdditionallyCP-870,893, a humanized IgG2 agonist antibody to CD40, showedencouraging activity in solid tumor indications when combined withpaclitaxel or carboplatin or gemcitabine. In these studies, activationof antigen presenting cells, cytokine production, and generation ofantigen-specific T cells were seen. See, e.g., Beatty et al., Clin.Cancer Res. 19:6286-6295 (2013) and Vonderheide et al., Oncoimmunology2:e23033 (2013).

Anti-CD40 Antibodies

Because of its role in immune function, antibodies have been raisedagainst the CD40 antigen. Such antibodies can be classified into threegroups, antagonistic antibodies, which inhibit CD40 activity; partiallyagonistic antibodies, which partially induce CD40 activity; and fullyagonistic antibodies, which fully stimulate CD40 activity. Members ofeach of the groups have been tested in clinical trials; none have beenapproved to date.

SEA-CD40

This disclosure provides a non-fucosylated hS2C6 antibody, SEA-CD40.S2C6 was originally isolated as a murine monoclonal antibody raisedagainst a human bladder carcinoma referred to herein as mS2C6. See,e.g., Paulie et al., Cancer Immunol. Immunother. 17:165-179 (1984). TheS2C6 antibody is a partial agonist of the CD40 signaling pathway andthus has the following activities: binding to human CD40 protein,binding to cynomolgus CD40 protein, activation of the CD40 signalingpathway, potentiation of the interaction of CD40 with its ligand, CD40L.See, e.g., U.S. Pat. No. 6,946,129.

As a next step in development, S2C6 was humanized and this humanizedantibody is referred to as humanized S2C6, herein, and alternatively asdacetuzumab, or fucosylated, humanized S2C6 (fhS2C6), or SGN-40. See,e.g., WO 2006/128103. SGN-40 was tested in human clinical trials and wasfound not to be sufficiently active to warrant further development.

SEA-CD40 is a non-fucosylated humanized S2C6 antibody. The amino acidsequences of the heavy and light chain for SEA-CD40 are disclosed as SEQID NO:1 and 2, respectively. The variable region of the heavy chain isfrom amino acids 1-113 of SEQ ID NO:1; the variable region of the lightchain is from amino acids 1-113 of SEQ ID NO:2. The generation of theantibody backbone of SEA-CD40 is disclosed at WO 2006/128103, which isherein incorporated by reference.

This disclosure provides a non-fucosylated, humanized S2C6 antibody,referred to herein as of hS2C6 or SEA-CD40. In addition to enhancedbinding to Fc receptors, SEA-CD40 also enhances activity of the CD40pathway, as compared to the parent antibody, dacetuzumab. The SEA-CD40antibody thus, is administered to patients at at lower doses and usingdifferent schedules of administration.

Non-Fucosylated Antibodies

SEA-CD40 is a non-fucosylated antibody and exhibits enhanced binding toFcγIII receptors, and surprisingly enhanced ability to activate the CD40signaling pathway in immune cells.

Methods of Making Non Fucosylated Antibodies

This disclosure provides compositions and methods for preparinghumanized S2C6 antibodies with reduced core fucosylation. As usedherein, “core fucosylation” refers to addition of fucose(“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducingterminal of an N-linked glycan.

Fucosylation of complex N-glycoside-linked sugar chains bound to the Fcregion (or domain) of the SEA-CD40 antibody backbone is reduced. As usedherein, a “complex N-glycoside-linked sugar chain” is typically bound toasparagine 297 (according to the EU index as set forth in Kabat,“Sequences of Immunological Interest, 5^(th) Ed., Pub. No. 91-3242, U.S.Dept. Health & Human Services, NIH, Bethesda, Md., 1991). As usedherein, the complex N-glycoside-linked sugar chain has a biantennarycomposite sugar chain, mainly having the following structure:

where ± indicates the sugar molecule can be present or absent, and thenumbers indicate the position of linkages between the sugar molecules.In the above structure, the sugar chain terminal which binds toasparagine is called a reducing terminal (at right), and the oppositeside is called a non-reducing terminal. Fucose is usually bound toN-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by anα1,6 bond (the 6-position of GlcNAc is linked to the 1-position offucose). “Gal” refers to galactose, and “Man” refers to mannose.

A “complex N-glycoside-linked sugar chain” includes 1) a complex type,in which the non-reducing terminal side of the core structure has one ormore branches of galactose-N-acetylglucosamine (also referred to as“gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAcoptionally has a sialic acid, bisecting N-acetylglucosamine or the like;or 2) a hybrid type, in which the non-reducing terminal side of the corestructure has both branches of a high mannose N-glycoside-linked sugarchain and complex N-glycoside-linked sugar chain.

In some embodiments, the “complex N-glycoside-linked sugar chain”includes a complex type in which the non-reducing terminal side of thecore structure has zero, one or more branches ofgalactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and thenon-reducing terminal side of Gal-GlcNAc optionally further has astructure such as a sialic acid, bisecting N-acetylglucosamine or thelike.

According to the present methods, typically only a minor amount offucose is incorporated into the complex N-glycoside-linked sugarchain(s) of the SEA-CD40 molecule. For example, in various embodiments,less than about 60%, less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, less than about 15%, less than about10%, less than about 5%, or less than about 3% of the antibody has corefucosylation by fucose. In some embodiments, about 2% of the antibodyhas core fucosylation by fucose.

In certain embodiments, only a minor amount of a fucose analog (or ametabolite or product of the fucose analog) is incorporated into thecomplex N-glycoside-linked sugar chain(s). For example, in variousembodiments, less than about 40%, less than about 30%, less than about20%, less than about 15%, less than about 10%, less than about 5%, orless than about 3% of the SEA-CD40 antibody has core fucosylation by afucose analog or a metabolite or product of the fucose analog. In someembodiments, about 2% of the SEA-CD40 antibody has core fucosylation bya fucose analog or a metabolite or product of the fucose analog.

Methods of making non-fucosylated antibodies by incubatingantibody-producing cells with a fucose analogue are described, e.g., inWO/2009/135181. Briefly, cells that have been engineered to express thehumanized S2C6 antibody are incubated in the presence of a fucoseanalogue or an intracellular metabolite or product of the fucose analog.As used herein, an intracellular metabolite can be, for example, aGDP-modified analog or a fully or partially de-esterified analog. Aproduct can be, for example, a fully or partially de-esterified analog.In some embodiments, a fucose analogue can inhibit an enzyme(s) in thefucose salvage pathway. For example, a fucose analog (or anintracellular metabolite or product of the fucose analog) can inhibitthe activity of fucokinase, or GDP-fucose-pyrophosphorylase. In someembodiments, a fucose analog (or an intracellular metabolite or productof the fucose analog) inhibits fucosyltransferase (preferably a1,6-fucosyltransferase, e.g., the FUT8 protein). In some embodiments, afucose analog (or an intracellular metabolite or product of the fucoseanalog) can inhibit the activity of an enzyme in the de novo syntheticpathway for fucose. For example, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity ofGDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In someembodiments, the fucose analog (or an intracellular metabolite orproduct of the fucose analog) can inhibit a fucose transporter (e.g.,GDP-fucose transporter).

In one embodiment, the fucose analogue is 2-flurofucose. Methods ofusing fucose analogues in growth medium and other fucose analogues aredisclosed, e.g., in WO/2009/135181, which is herein incorporated byreference.

Other methods for engineering cell lines to reduce core fucosylationincluded gene knock-outs, gene knock-ins and RNA interference (RNAi). Ingene knock-outs, the gene encoding FUT8 (alpha 1,6-fucosyltransferaseenzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residuefrom GDP-fucose to position 6 of Asn-linked (N-linked) GlcNac of anN-glycan. FUT8 is reported to be the only enzyme responsible for addingfucose to the N-linked biantennary carbohydrate at Asn297. Geneknock-ins add genes encoding enzymes such as GNTIII or a golgi alphamannosidase II. An increase in the levels of such enzymes in cellsdiverts monoclonal antibodies from the fucosylation pathway (leading todecreased core fucosylation), and having increased amount of bisectingN-acetylglucosamines. RNAi typically also targets FUT8 gene expression,leading to decreased mRNA transcript levels or knocking out geneexpression entirely. Any of these methods can be used to generate a cellline that would be able to produce a non-fucosylated antibody, e.g., anSEA-CD40 antibody.

Those of skill will recognize that many methods are available todetermine the amount of fucosylation on an antibody. Methods include,e.g., LC-MS via PLRP-S chromatography and electrospray ionizationquadrupole TOF MS.

The non-fucosylated antibody, SEA-CD40, when adminstered to a patientinduces activation of monocyte maturation into macrophages and induceproduction of cytokines, including, e.g., interferon-γ (IFN-γ) andchemokine that elicit robust T-cell response to immune systemchallenges. Unlike fully agoninstic antibodies, such as antibody 24.4.1,SEA-CD40 does not induce production of immune-dampening cytokines, suchas interleukin-10 (IL-10). IL-10, in turn, induces activity ofT-regulatory cells, which dampen the immune response. Thus, SEA-CD40 isuseful for induction of a robust T-cell mediated immune response withoutpromoting activity of T-regulatory cells.

Dosage and Administration of SEA-CD40

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically-compatible buffers to reduce discomfort at the siteof injection. The solution can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternativelyantibodies can be in lyophilized form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

SEA-CD40 is administered intravenously. In other embodiments, SEA-CD40is administered subcutaneously. In a further embodiment, SEA-CD40 isadministered subcutaneously at the site of a tumor.

The non-fucosylated SEA-CD40 antibody has surprisingly enhanced immuneactivation activity as compared to its parent antibody, dacetuzumab.Thus, SEA-CD40 can be administered to patients at lower doses and ondifferent schedules as compared to dacetuzumab.

As an example, SEA-CD40 can be adminstered to patients at levels between0.1-2000 μg/kg (μg antibody per kilogram patient body weight). Otherpossible dosage ranges are 10-1000 μg/kg, 50-800 μg/kg, 75-600 μg/kg,100-500 μg/kg. Other possible dosage ranges are the following: 100-300μg/kg, 300-500 μg/kg, 500-700 μg/kg, 700-900 μg/kg, and 900-1100 μg/kg.Still more dose ranges are the following: 100-150 μg/kg, 150-200 μg/kg,200-250 μg/kg, 250-300 μg/kg, 300-350 μg/kg, 350-400 μg/kg, 400-450μg/kg, 450-500 μg/kg, 500-550 μg/kg, 550-600 μg/kg, 600-650 μg/kg,650-700 μg/kg, 700-750 μg/kg, 750-800 μg/kg, 800-850 μg/kg, 850-900μg/kg, 900-950 μg/kg, 950-1000 μg/kg, 1000-1050 μg/kg, and 1050-1100μg/kg. Other possible dosage ranges are 0.3-200 μg/kg, 0.6-150 μg/kg,1.0-100 μg/kg, 2-50 μg/kg, 5-25 μg/kg, 7.5-15 μg/kg, and 8-12 μg/kg.

In other embodiments, SEA-CD40 is administered to patients at 0.6 μg/kg,1.0 μg/kg, 2.5 μg/kg, 5.0 μg/kg, 7.5 μg/kg, 10 μg/kg, 30 μg/kg, 50μg/kg, 75 μg/kg, 100 μg/kg, or 200 μg/kg. In a preferred embodiment,SEA-CD40 is administered to patients at 10 μg/kg.

In further embodiments, SEA-CD40 is administered to patients at about 60μg/kg, about 100 μg/kg, about 150 μg/kg, about 200 μg/kg, about 250μg/kg, about 300 μg/kg, about 350 μg/kg, about 400 μg/kg, about 450μg/kg, about 500 μg/kg, about 550 μg/kg, about 600 μg/kg, about 650μg/kg, about 700 μg/kg, about 750 μg/kg, about 800 μg/kg, about 850μg/kg, about 900 μg/kg, about 950 μg/kg, about 1000-1050 μg/kg, about1050 μg/kg, and 1110 μg/kg.

In some embodiments, SEA-CD40 is administered in a manner to reduce thelikelihood of immune exhaustion. For example, SEA-CD40 can beadministered at three week intervals, six week intervals, eight weekintervals, ten week intervals, twelve week intervals, or 14 weekintervals. Intervals can also be on a monthly schedule, e.g., one monthintervals, two month intervals, or three month intervals.

Because SEA-CD40 activates the immune system to respond againsttumor-related antigens, its use is not limited to cancers that expressCD40. Thus SEA-CD40 can be used to treat both CD40 positive and CD40negative cancers.

SEA-CD40 is preferably used to treat tumors that are known to be immuneresponsive, particularly if the cancer expresses low levels of CD40 ordoes not detectably express CD40. Immune responsive cancers include,e.g., melanoma; bladder cancer; lung cancer, e.g., small cell lungcancer and non-small cell lung cancer; ovarian cancer; kidney cancer;pancreatic cancer; breast cancer; cervical cancer; head and neck cancer,prostate cancer; glioblastoma; non-hodgkin lymphoma; chronic lymphocyticleukemia; hepatocellular carcinoma; or multiple myeloma.

In another embodiment, SEA-CD40 is used to treat solid tumors. In afurther embodiment, SEA-CD40 is used to treat blood cancers, e.g.,lymphoma, including non-Hodgkin lymphoma and Hodgkin lymphoma; chroniclymphocytic leukemia; or multiple myeloma.

SEA-CD40 Combination Therapy

Because of its immune stimulatory function, SEA-CD40 can be used incombination with other therapeutic agents that activate the immunesystem. Drugs with immune stimulatory function include, e.g., T-cellmodulators, including immune checkpoint inhibitors; immune activators;and chemotherapeutic agents that induce immunogenic cell death. As anexample, certain antibodies function by blocking activity of moleculesthat serve as immune checkpoints on T cells. SEA-CD40 can, therefore beused in combination with antibodies that target immune checkpointproteins.

T-Cell Modulators

T-cells play a role in the ability of the immune system to recognize andeliminate cancers from the body. T-cell modulators include antibodiesthat block the function of immune checkpoints. See, e.g., Pardoll,Nature Rev. Cancer, 12:252-264 (2012). Antibodies that block immunecheckpoints include, e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies,and anti-CTLA4 antibodies. Other checkpoint inhibitors/activatorsinclude LAG3 and TIM3. Antibodies against some proteins can be used tomodulate T-cell activity or preferably activate T-cell activity, e.g.,antibodies against 41BB, CD27, ICOS, and OX40. Other T-cell modulatorsinclude inhibitors of the enzyme indolamine 2,3-dioxygenase (DO).

Anti-CTLA4 antibodies recognize the protein cytotoxic lymphocyte 4(CTLA-4), also known as cluster of differentiation 152 or CD152. TheCTLA-4 protein is expressed on T cells, which recognize antigens thatare suitable for attack by the immune system. Activation of CTLA-4dampens the immune response. See e.g., Nirschi and Drake, Clin. CancerRes., 19:4917-4924 (2013). Antibodies specific for CTLA-4 and that blockits activity have been used to treat cancer by upregulating the immuneresponse to cancers. Examples of CTLA-4 antibodies include ipilimumab ortremelimumab. SEA-CD40 can be administered in combination withipilimumab or tremelimumab to treat cancer.

Anti-PD1 antibodies recognize the protein programmed death-1 (PD-1).Like CTLA-4, PD-1 is expressed on T cells, and dampens the immuneresponse. See e.g., Nirschi and Drake, Clin. Cancer Res., 19:4917-4924(2013). Antibodies specific for PD-1 and that block its activity havebeen used to treat cancer by upregulating the immune response tocancers. Examples of PD-1 antibodies include MEDI0680, AMP-224,nivolumab, pembrolizumab, and pidilizumab. Other PD-1 binding proteinsthat act as checkpoint inhibitors and can be used in combination withSEA-CD40 include, e.g., B7-DC-Fc. SEA-CD40 can be administered incombination with MEDI0680, AMP-224, nivolumab, pembrolizumab, orpidilizumab to treat cancer.

PD-L1 is a ligand of the PD-1 protein. PD-L1 is expressed on cancercells and its interaction with PD-1 allows PD-L1-expressing cancer cellsto evade the immune system. Anti-PD-L1 antibodies have been generatedand used to treat cancer. Examples of PD-L1 antibodies include, e.g.,MEDI4736, BMS-936559/MDX-1105, MSB0010718C and MPDL3280A. SEA-CD40 canbe administered in combination with MEDI4736, BMS-936559/MDX-1105,MSB0010718C or MPDL3280A to treat cancer.

Other antibodies that block the function of immune checkpoint proteinsinclude antibodies directed against e.g., LAG3 and TIM3, and can be usedin combination with SEA-CD40.

Antibodies against 41BB, CD27, ICOS, and OX40 are used to activateT-cell activity and can be used in combination with SEA-CD40. OX40antibodies include, e.g., MEDI6469 and MEDI6383. An example of anagonistic anti-CD27 antibody is CDX-1127, which can be used incombination with SEA-CD40.

The enzyme indolamine 2,3-dioxygenase (DO) catalyzes the degradation ofthe amino acid tryptophan. Inhibitors of DO can be small molecules, suchas rosmarinic acid, COX-2 inhibitors, and alpha-methyl-tryptophan.

Chemotherapeutic Agents that Induce Immunogenic Cell Death

In most humans, millions of cells die via apoptosis and are removedwithout generating an immune response. However, after treatment withsome chemotherapeutic agents, immune cells have been observed toinfiltrate tumors. Thus, some tumor cells killed by chemotherapeuticagents act as vaccines and raise a tumor-specific immune response. Thisphenomenon is referred to as immunogenic cell death (ICD). See, e.g.,Kroemer et al., Annu. Rev. Immunol., 31:51-72 (2013). The ability of achemotherapeutic agent to induce ICD can be determined experimentally.Two criteria must be met. First, injection of an immunocompetent mousewith cancer cells that have been treated in vitro with achemotherapeutic agent must elicit a protective immune response that isspecific for tumor antigens, in the absence of adjuvant. Second, ICDoccurring in vivo, e.g., a mouse syngeneic model with treatment using apotential ICD-inducing chemotherapeutic agent, must drive an immuneresponse in the tumor that is dependent on the immune system.

Chemotherapeutic agents that induce ICD include, e.g., anthracyclines,anti-EGFR antibodies, bortezomib, cyclophosphamide, gemcitabine,irradiation of the tumor, and oxaliplatin. SEA-CD40 can be used incombination with any of these agents to generate an enhanced immuneresponse and treat cancer in a patient.

Immune Activation

Cancer can is also treated by administering agents that directlystimulate the immune system. Such agents include, e.g., GM-CSF,IFN-gamma, interleukin-2, GVAX, and TLR9 agonists. Other immuneactivators include, e.g., cancer vaccines, Bacillus Calmette-Guérin(BCG), nonspecific immunostimulants (e.g. imiquimod) and cellulartherapies like CAR-T cells. SEA-CD40 can be used in combination with anyof these agents to generate an enhanced immune response and treat cancerin a patient.

Other Combinations

Other combinations with SEA-CD40 can be used to treat cancer. Examplesinclude, e.g., SEA-CD 40 in combination with an anti-PD1 antibody, e.g.,nivolumab, pembrolizumab, and pidilizumab, MEDI0680, or AMP-224;SEA-CD40 in combination with Gemcitabine, with or without paclitaxel orcisplatin or oxaliplatin; SEA-CD-40 in combination with a BRAFinhibitor, e.g., vemurafenib or dabrafenib; or SEA-CD40 in combinationwith cyclophosphamide, ADRIAMYCIN™, vincristine, and prednisone (CHOP)or rituximab, ifosfamide, carboplatin, and etopiside (RICE) orrituximab, gemcitabine, dexamethasone and cisplatin (RGDP).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Synthesis of Non-Fucosylated hS2C6 Antibody

The humanized anti-CD40 antibody, S2C6 with heavy and light light chainsof SEQ ID NOs: 1 and 2 was expressed in CHO cells. A fucosylationinhibitor, 2-fluorofucose, was included in the cell culture media duringthe production of antibodies resulted in non-fucosylated antibody,SEA-CD40. See, e.g., Okeley et al., Proc. Nat'l Acad. Sci.110:5404-55409 (2013). The base media for cell growth was fucose freeand 2-flurofucose was added to the media to inhibit proteinfucosylation. Ibid. Incorporation of fucose into antibodies was measuredby LC-MS via PLRP-S chromatography and electrospray ionization quadropleTOF MS. Ibid. Data not shown.

Example 2: Characterization of Non-Fucosylated hS2C6 Antibody

CD40 Binding affinity determination of SEA-CD40: For isolation ofperipheral blood mononuclear cells (PBMCs), human whole blood wassupplied by ASTARTE BIOLOGICS™. Briefly, blood was collected intoheparin tubes and delivered to SEATTLE GENETICS™ within four hours ofbeing drawn. Upon arrival blood was aliquoted into 50 ml conical tubes(falcon) and spun at 200 g in an EPPENDORF™ 5810R (A-4-62 rotor) for 20minutes at 25° C., without break to separate the platelet rich fraction.Following centrifugation, three distinct layers were formed: bottomlayer, red blood cells (accounting for 50-80% of the total volume);middle layer, very thin band of white blood cells; top layer,straw-colored platelet rich plasma (PRP).

The upper straw colored layer with which is enriched in platelets wasremoved with a one ml pipette. Once the platelet rich plasma was removedblood was diluted with equal volumes of sterile PBS (GIBCO™, lot1618435, ept 2016-07). 15 mls of HISTOPAQUE™-1077 (SIGMA™, lot numberRNBD2965, Expt. 5/2017) warmed to room temperature was underlayeredbelow the blood. HISTOPAQUE™ samples were spun at 1500 rpm for 25minutes at 25° C. with outbreak. Following centrifugation three layersare formed again: bottom layer, red blood cells (accounting for 50-80%of the total volume); middle layer, thick band of white blood cells(also called “buffy coat”); top layer, PBS and remaining platelets.

The upper PBS/Platelet layer was removed with a 1 ml pipet anddiscarded. The thick band of white blood cells was gently removed andplaced into a clean 50 ml sterile conical tube. Tubes were filled to 50mls and cells are spun at 800 g for 10 minutes. Wash solution wasremoved and pellets were resuspended in 10 mls of ACK red blood lysisbuffer (GIBCO™, lot 1618488) for ten minutes. Fifty milliliter conicaltubes were then topped off with 35 ml sterile PBS and cells were spun at800 g for ten minutes. The wash solution was removed and pellet wasresuspended in 50 mls of PBS. Five hundred μl of sample was removed andPBMC were counted with a Vi-cell-XR (BECKMAN COULTER™). Cells were spunagain at 800 g for ten minutes. The wash solution was removed and pelletre-suspended at 1×10⁶/ml in FACs staining solution (BD). One hundred μlof resuspended PBMC's were plated into a 96 well U-bottom plate(CORNING™) and placed on ice. To block non-specific FcγRIIIa binding,PBMC's were pre-treated 100 μg/ml of human Fc-fragments (CALBIOCHEM™,)for thirty minutes. Ten-fold serial dilutions of biotinylated SEA-h00(non-fucosylated control antibody), SEA-CD40, or SGN-40 were prepared tocreate a dilution series of (100, 10, 1, 0.1, 0.01, 0.001, 0.0001μg/ml).

Samples were washed twice in ice cold FACs buffer and incubated withsaturating concentrations of PE-Streptavidin (BD™) on ice for thirtyminutes. Samples were washed twice in ice cold FACs buffer andre-suspended in 200 μl of FAC's buffer. Binding was assessed using a BDLSRII and DIVA software. FCS were analyzed in FLOWJO™ and GeoMeanfluorescence of positively stained cells was determined and plotted inPrism Graph Pad. Data was fit to non-linear regression assuming onebinding site in Prism and binding K_(D) values calculated by dividingμg/ml calculation by molecular weight of SEA-CD40.

Results: The binding affinity of SEA-CD40, and the parental antibodydacetuzumab, to CD40 on human peripheral blood mononuclear cells (PBMC)was determined by flow cytometry. Background binding of an appropriateisotype control was subtracted and mean fluorescence intensity (MFI) wasplotted against antibody concentration. Results are shown in FIG. 1 .SEA-CD40 and the parental antibody dacetuzumab gave virtuallyoverlapping binding curves and both saturated PBMC's at concentrationsof approximately 1.17 nM. These data suggest that changes infucosylation do not affect SEA CD40 affinity for CD40.

FcγRIIIa Binding affinity determination of SEA-CD40: CHO cells thatexpress the high (158V) or low (158F) version of human FcγRIIIa weregenerated. 20×10⁶ cells were centrifuged, washed once in 20 ml 1×PBS,and resuspended in 8 ml BD stain buffer. Cells were aliquoted in thefollowing density: 2.0×10⁶ cells/ml in 100 μl volume. 0.20×10⁶ cellswere aliquoted to each well. Cells were centrifuged at 1250 rpm, forfive minutes at room temperature. Antibodies were diluted to either 0.14ug/ml (SGN) or 0.04 ug/ml (SEA). Dilutions are provided in Table 1.

TABLE 1 Biotinylated abs Conc. Vol (ul) Vol. stain Highest staindilutions Mg/ml antibody buffer cone ug/ml SGN-40-Biotin 3.29 18.23581.7 100 SEA40-Biotin 3.27 15.11 584.7 100 h00-SGN-Biotin 1.55 38.7561.0 100 h00-SEA biotin 3.61 16.6 583 100Supernatants were aspirated from the spun cells and 60 μl ofcorresponding antibody dilutions were added with a multichannel pipet.Corresponding concentrations were 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.14mcg/ml. Samples were incubated at 4° C. for 1 hour. Samples werecentrifuged, and washed twice with 200 μl BD stain buffer per well. Onemilliliter of Streptavidin-PE was added to 20 ml BD™ stain buffer(excess 2°) to make streptavidin buffer. 100 μl of streptavidin bufferwas added to each sample and they were incubated for 30 min in the darkat 4° C. Samples were then centrifuged and washed twice with 200 ul BD™Stain buffer per well. Samples were analyzed by Flow cytometry in HTSmode on the LSRII and graph MFI to calculate Kd's in PRIZM.

Results: Binding of SEA-CD40 and the parent antibody dacetuzumab toChinese hamster ovary (CHO) cells expressing the low (158F) or high(158V) affinity form of FcγRIIIa was assessed. Results are shown inFIGS. 2A and 2B. SEA-CD40 bound to both the low (158F) and high (158V)form of FcγRIIIa with similar affinity (K_(D) 27.5 nM and 5.2 nM,respectively). SEA-CD40 binding to the low affinity (158F) form wassignificantly better than the fucosylated parental antibody dacetuzumab(K_(D) 302.7 nM), and SEA-CD40 even bound the high affinity 158V formbetter than the fucosylated dacetuzumab parent (5.2 nM vs. 37.9 nMrespectively).

SEA-CD40 mediated ADCC activity: Human PBMC's were isolated as above andwere treated with various concentrations of SEA-CD40 or an SEA-isotypecontrol (SEA-h00) for 6, 24, or 48 hours. Cultures were stained forCD19+ B cells and cell numbers were quantified by flow cytometry.

Results: Human PBMC cultures, were treated with 100, 10, 1, 0.1, 0.01,0.001, or 0.0001 μg/mL of SEA CD40 or a non-binding SEA-isotype control(SEA-h00) for 6, 24, and 48 hours and the number of CD40 positive cellswere then assessed. Results are shown in FIG. 4 . SEA-CD40 treatmentresulted in a significant decrease in CD40+ CD19+ B cells in a dose- andtime-dependent manner, even down to low sub μg/mL concentrations. Therewas no significant effect of SEA-CD40 on monocyte/DC numbers(monocyte/DC data not shown).

Assessment of cytokine production in human whole blood or PBMCs: Humanwhole blood was supplied by ASTARTE BIOLOGICS™. Briefly, 100 mls ofblood was collected into heparin tubes and delivered to SEATTLEGENETICS™ within 4 hours of the draw. Half of the blood was set asidefor whole blood cultures while the other half was used to isolate PBMCsas described above. One hundred μl of whole blood was aliquoted into3-96 flat bottom tissue culture plates (COSTAR™). Isolated PBMCs werecounted in a VIACELL™ and resuspended at 1×10⁶ cells/ml in DMEMcontaining 10% FBS (ASTARTE BIOLOGICS™), 1× penicillin/strepA, and Xglutamine (PBMC media). For PBMCs, one hundred μl of resuspened,purified PBMCs were aliquoted into 3-96 flat bottom tissue cultureplates. 10× serial dilutions of SEA-h00 and SEA-CD40 were made in PBMCmedia and whole blood and isolated PBMC cultures were treated withdescending concentrations of either SEA-h00 or SEA-CD40 (100, 10, 1.0,0.1, 0.01, 0.001, 0.0001 or 0 μg/ml). SEA-CD40 treatment was performedin duplicate for both whole blood and PBMC cultures at each time point.At each of the pre-determined times points (6, 24, and 48 hours) a 96well plate containing whole blood or purified PBMCs was spun with aplate adapter in an EPPENDORF™ 5810R at 800 rpm for 5 minutes. Serum ortissue culture supernatants were removed and transferred to a 96 striptube rack and samples were frozen at −80° C. until processing.

Frozen tissue culture supernatants and serum were thawed overnight at 4°C. and processed for cytokine production using a LUMINEX™ multiplex Kitfrom MILLIPORE™ Custom kits were designed to analyze IFNγ, IL-12p40,IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TNF-α, sCD40L. Analytes were pickedbased on cytokines observed with dacetuzemab in previous studies. Tissueculture supernatants and serum samples were processed as per themanufactures instructions. Briefly, assay plates were washed with 200 μLof wash buffer per well, followed by addition of 25 μL standard orbuffer, 25 μL matrix or sample, and 25 μL of multiplexed analyte beadsto each well. Samples were incubated overnight with vigorous shaking at4° C. Plates were washed twice with wash buffer. Twenty-five μL ofdetection antibodies were added to each well and incubated at roomtemperature for one hour. Twenty-five μL of streptavidin-phycoerythrin(SA-PE) were added and samples incubated at room temperature for thirtyminutes. The plate was washed twice with wash buffer and beads wereresuspended with 150 μL of sheath fluid. The samples were analyzed usingLUMINEX™ MAGPIX™ systems in combination with the XPONENT™ softwaresystem. Cytokine levels were calculated from the standard curve.

Results: Human whole blood cultures were treated with a SEA-isotypecontrol or SEA-CD40 (100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 μg/mL) for6, 24, or 48 hours. Serum or tissue culture supernatants were collectedand inflammatory cytokines assessed by multiplexed LUMINEX™ analysis.The data are plotted as a fold increase in cytokine production comparedto a SEA-isotype control. SEA-CD40 stimulated robust production of IFNγ,MIP1β, and TNFα at 6, 24, and 48 hours in whole blood, as shown in Table2, below. SEA-CD40 levels are provided in the leftmost columns. Activitywas observed at levels as low as 0.010 μg/mL SEA-CD40. Stimulation ofMIP1β, and TNFα at twenty-four hours are shown in FIGS. 4A and 4B.

TABLE 2 Whole Blood 6 hrs IFNγ IL-8 MCP-1 MIP1α MIP1β TNFα 100.00 4.522.01 2.75 1.30 30.86 3.22 10.00 10.81 1.05 2.33 1.04 26.69 1.90 1.004.99 1.13 1.62 0.93 4.59 2.20 0.10 3.42 0.84 0.96 1.02 0.88 1.65 0.011.83 1.00 1.29 1.26 0.96 0.98 0.00 1.20 0.94 1.25 1.24 0.93 1.04 0.001.16 1.05 1.29 1.15 0.98 1.02 24 hr IFNγ IL-8 MCP-1 MIP1α MIP1β TNFα100.00 3.01 1.95 2.19 2.28 6.77 3.51 10.00 3.23 1.52 2.84 2.34 8.42 3.261.00 2.31 1.70 2.36 1.75 6.77 3.62 0.10 1.32 1.36 1.19 0.89 3.95 2.120.01 0.95 1.10 1.01 0.74 0.96 2.11 0.00 0.55 0.92 1.04 0.95 1.15 1.030.00 0.40 0.82 0.79 1.01 1.66 1.44 48 hrs IFNγ IL-8 MCP-1 MIP1α MIP1βTNFα 100.00 3.59 1.11 1.19 1.26 2.03 3.47 10.00 2.37 1.21 1.22 1.24 2.272.71 1.00 2.15 1.08 1.07 1.07 1.76 2.63 0.10 1.01 0.76 1.05 1.09 1.432.53 0.01 0.86 0.81 1.16 1.17 1.27 1.93 0.00 0.87 0.97 1.18 1.18 0.971.18 0.00 0.96 0.93 0.87 1.05 0.68 1.17

Human PBMC were treated with a SEA-isotype control or SEA-CD40 (100, 10,1, 0.1, 0.01, 0.001, or 0.0001 μg/mL) for 6, 24, or 48 hours. Serum ortissue culture supernatants were collected and inflammatory cytokinesassessed by multiplexed LUMINEX™ analysis. The data are plotted as afold increase in cytokine production compared to a SEA-isotype control.SEA-CD40 stimulated robust production of IFNγ, MIP1β, and TNFα at 6, 24,and 48 hours PBMCs, as shown in Table 3, below. SEA-CD40 levels, inμg/mL, are provided in the leftmost columns. Activity was observed atlevels as low as 0.010 μg/mL SEA-CD40. Stimulation of MIP1β, and TNFα attwenty-four hours are shown in FIGS. 5A and 5B.

TABLE 3 PBMC 6 hr IFNγ IL-8 MCP-1 MIP1α MIP1β TNFα 100 8.75 1.18 3.002.51 6.91 11.66 10 13.68 1.16 7.70 3.11 11.59 17.72 1 6.21 0.89 2.711.48 4.55 5.58 0.1 3.89 0.89 1.61 1.26 3.40 3.04 0.01 1.49 0.75 1.071.26 2.11 2.26 0.001 1.60 0.71 0.89 1.31 1.30 1.28 0.0001 1.58 0.71 0.770.83 1.33 1.10 24 hr IFNγ IL-8 MCP-1 MIP1α MIP1β TNFα 100 8.51 4.79 5.692.84 13.43 19.91 10 8.98 3.96 7.87 1.91 7.58 14.97 1 3.32 1.35 3.10 1.288.73 3.79 0.1 1.80 1.04 1.38 1.03 5.85 5.58 0.01 1.66 0.85 1.28 1.082.87 1.22 0.001 1.12 0.71 0.90 0.96 1.18 2.75 0.0001 0.40 0.71 0.80 0.741.11 1.02 48 hrs IFNγ IL-8 MCP-1 MIP1α MIP1β TNFα 100 17.92 2.58 11.471.51 2.81 14.02 10 8.81 3.61 3.39 1.46 2.33 5.58 1 4.09 2.07 2.36 1.321.91 6.47 0.1 1.82 1.19 0.84 0.96 1.00 2.30 0.01 1.03 1.02 1.41 0.951.13 2.09 0.001 0.83 0.86 1.13 0.93 0.96 1.93 0.0001 0.82 0.97 0.97 0.911.05 1.73

Assessment of Activation markers on PBMCs: Co-stimulatory moleculesurface expression was assessed on the cell pellets remaining from thecytokine analysis described above. Cell pellets were resuspended in 50ml of BD FACs buffer and transferred to and 96 well round bottomedmicrotiter plates Fc receptors were blocked with human 100 μg/mlFc-fragments (MILLIPORE™) for 30 minutes on ice. A master mix composedof PE-CD86 (BD) and MHCII (Pan anti-DR,DP,DQ antibody BD) diluted at1:100 was prepared in BD FACs buffer containing 100 mg/ml human Fcfragments. Five μl of the master mix was added to each well containingninety μl and samples were incubated for one hour on ice. Cells werethen spun at 400 g in a pre-cooled EPPENDORF™ 5810R centrifuge for fiveminutes. Supernatants were removed and cells washed with 200 ml of BDFACs buffer. Cells were washed twice and then resuspended in 200 ml ofFACs buffer. Samples were then analyzed on an LSRII (BD™ biosciences)with DIVA software (BD™ biosciences). CD86 and MHCII geo meanfluorescence was assessed using FLOWJO™ analysis software. A ratiobetween SEA-h00 and SEA-CD40 was calculated and the fold change used tocalculate a range of SEA-CD40 potency.

Results: Activation of CD40, in addition to eliciting cytokineproduction, promotes the maturation of antigen presenting cells. DCmaturation can be followed by upregulation of activation markersincluding CD86 and MHCII. Human PBMC cultures were stimulated with SEACD40 and an SEA-isotype control for 6, 24, or 48 hours and surfaceexpression of MHC Class II antigens (HLA DR, DP, DQ) and CD86 wasassessed. SEA-CD40 stimulation, but not the isotype control resulted ina significant increase in both MHCII (Table 4) and CD86 (data not shown)at concentrations as low as 0.01 μg/mL.

TABLE 4 SEA- MHCII CD40 6 hrs 24 hrs 48 hrs 100 1.13 1.64 2.10 10 1.101.62 2.10 1 1.14 1.76 1.57 0.1 0.90 1.19 1.50 0.01 0.90 1.21 1.38 0.0011.00 1.07 1.10 0.0001 0.85 1.05 0.99

Role of fucose in immune activation by SEA-CD40: Human PBMCs wereisolated as described above. PBMCs were treated with variousconcentrations of SEA-CD40, the parental antibody SGN40, or a F(ab)′2version of SEA-CD40 and incubated for 24 hrs. For assessment of B celldepletion, BPMC cultures were stained with a PE-CD19 and B-cell numberswere quantified by flow cytometry. For assessment of cytokineproduction, PBMC tissue culture supernatants were collected and cytokineproduction assessed by multiplex analysis on the Luminex platform. IFNγproduction is shown is shown in FIG. 7 , (ng/mL) and similar trends wereseen for the other cytokines. For assessment of antigen presenting cellmaturation, PBMC's were collected after the twenty-four hour incubationand stained with aPE-anti-CD86 or APC-pan MHC Class II antigen (DR, DQ,DP) antibody and the percent positive cells were assessed by flowcytometry. The data are shown as mean fluorescent intensity for pan MHCmarkers.

Results: To assess B cell depletion, human PBMC cultures were treatedwith multiple concentrations of SEA-CD40, dacetuzumab, or SEA-CD40F(ab′)2 for twenty-four hours. Results are shown in FIG. 6 . TheADCC-mediated depletion of B cells was significantly higher inSEA-CD40-treated cultures compared with dacetuzumab-treated cultures andthis activity was lost with the SEA-CD40 F(ab)′2.

Additionally immune activation endpoints (cytokines and APCactivation/maturation markers) were assessed in SEA-CD40, dacetuzumab,and SEA-CD40 F(ab′)2 PBMC cultures stimulated for twenty-four hours.SEA-CD40 stimulation of both cytokine production (FIG. 7 ) and APCmaturation (FIG. 8 ) was significantly higher than that of dacetuzumabor SEA-CD40 F(ab)′2. These data demonstrate that the lack of fucose onthe IgG domain does not alter CD40 binding, but does increase FcγRIIIabinding resulting in increased CD40 activity and ultimately increasedCD40 immune modulatory activity.

Example 3: Immune Modulatory Activity of Non-Fucosylated hS2C6 Antibody

Identification of active doses of SEA-CD40: SEA-CD40 is proposed to beactive at dose levels that activate antigen-presenting cells, which canbe characterized by upregulation of activation markers, such as MHCclass I or II, or CD86. Activation markers on PBMCs following treatmentwith various concentrations of SEA-CD40 from 6 to 48 hours were assessedas described above (Assessment of Activation markers on PBMCs). Thedifference between treatments with isotype control SEA-h00 and SEA-CD40for each activation marker were calculated and plotted versus SEA-CD40concentrations and treatment time. The steepest response-concentrationcurves were observed at 24 hours for CD86 and MHCII and 48 hours forMHCI. Response-concentration data (24 hr CD86, 24 hr WWII and 48 hr WWI)were fitted by nonlinear regression using the following equation, where0% and 100% responses are defined as the smallest and largest values inthe data set for each activation marker. EC50 is the concentration ofSEA-CD40 that gives a 50% response.

${Response} = \frac{100}{1 + {10^{({{\log_{10}{EC}50} - {\log_{10}{Concentration}}})}}}$

Results: Normalized response versus−log (concentration) and nonlinearfitted regression lines for activation markers are illustrated in FIG. 9. Estimated EC50 values for MHCI, CD86 and MHCII were 0.011, 0.14 and0.41 μg/mL, respectively. SEA-CD40 is estimated to induce approximately90%, 60%, and 30% maximal upregulation of MHCI, CD86 and MHCII,respectively, at 0.2 μg/mL, corresponding to a theoretical plasma Cmaxachievable by an IV dose of 10 μg/kg in humans. This dose is proposed asthe theoretical first anticipated active dose.

Example 4: Immune Modulatory Activity of Non-Fucosylated hS2C6 Antibody

T-cell response generated by SEA-CD40: An anti-M1 T cell line wasgenerated at ASTARTE BIOLOGICS™ from a HLA-A2 donor that was shown to behighly reactive to the M1 flu peptide. These cells were labeled withcarboxyfluorescein succinimidyl ester (CFSE) and combined withautologous PBMCs. Cultures were stimulated with 10 μg/ml M1 flu peptidein the presence or absence of decreasing concentrations (1, 0.1, 0.01μg/ml) of SEA-CD40 or dacetuzumab for 5 days. Cultures supernatants werecollected and analyzed for cytokines by multiplex analysis on theLUMINEX™ platform and antigen specific T-cells were identified by M1specific Tetramer binding.

Results: PBMC's from an HLA-A2 donor shown to be highly reactive to theM1 flu peptide were stimulated with M1 flu peptide in the presence orabsence of decreasing concentrations of SEA-CD40 or dacetuzumab for fivedays. Results are shown in FIGS. 10A and 10B. SEA-CD40 stimulatedcultures showed increased response to M1 flu antigen as seen by anincrease in IFNγ production and an increase in antigen specific T-cellsas determined by increased Tetramer binding. SEA-CD40 stimulated anantigen specific T-cell response down to 0.1 ug/ml and this activity wasmore robust than the response associated with dacetuzumab.

T-cell response generated by SEA-CD40 in combination with anti-immunecheckpoint inhibitor antibodies: An anti-M1 T cell line was generated atASTARTE BIOLOGICS™ from a HLA-A2 donor that was shown to be highlyreactive to the M1 flu peptide. These cells were labeled with CFSE andcombined with autologous PBMCs. Cultures were stimulated with 10 μg/mlM1 flu peptide in the presence or absence of decreasing concentrations(1, 0.1, 0.01 μg/ml) of SEA-CD40 and/or 1 μg/ml of an anti-CTLA4 or ananti-PD-1 antibody for five days. Culture supernatants were collectedand analyzed for cytokines by multiplex analysis on the LUMINEX™platform and antigen specific T-cells were identified by M1 specificTetramer binding.

Results: PBMC's from an HLA-A2 donor shown to be highly reactive to theM1 flu peptide were stimulated with M1 flu peptide in the presence orabsence of decreasing concentrations of SEA-CD40 and/or a constantconcentration of an anti-CTLA4 blocking antibody or an anti-PD-1antibody. Results are shown in FIGS. 11 and 12 . While SEA-CD40,anti-CTLA4 antibodies and anti-PD-1 antibodies alone stimulated anantigen specific T-cell response, increased response to M1 flu antigenas seen by an increase in IFN-γ production (FIG. 11 ) and an increase inantigen specific T-cells as determined by increased Tetramer binding(FIG. 12 , using the Tetramer/APC-HLA-A*02:01 Influenza-M1 (GILGFVFTL)tetramer from MBL) was observed when SEA-CD40 and anti-CTLA4 antibodiesor anti-PD-1 antibodies were combined. SEA-CD40 stimulated an antigenspecific T-cell response down to 0.1 ug/ml and this activity wasenhanced by combination with an immune checkpoint antibody.

T-cell response generated by SEA-CD40 in PCMCs from cancer patients: Forassessment of anti-CD40 antibodies alone, PBMCs were isolated from 10mls of tumor patient blood and 0.25 million cells were plated in a 24well plate. Samples were treated with increasing concentrations ofSEA-CD40 only, or 1 ug/ml of a combined peptide pool containingMageA1/MageA3/NY-ESO and increasing concentration of either SEA-CD40 orSGN-40. Samples were cultured in 10% CO₂ at 37° C. for five days, tissueculture supernatants were collected and INF-γ levels were assessed.

For assessment of SEA-CD40 in combination with immune checkpointblocking antibodies, PBMCs were isolated from 10 mls of blood frompatients diagnosed with either breast, pancreatic, of melanoma cancer,and 0.25 million cells were plated in a 24 well plate. Samples weretreated with increasing concentrations of SEA-CD40, 1 μg/ml of acombined peptide pool containing MageA1/MageA3/NY-ESO, and either 1μg/ml of anti-PD1 or anti-CTLA4. Samples were cultured in 10% CO₂ at 37°C. for 5 days, tissue culture supernatants were collected and INF-γlevels were assessed.

Results: PBMC's from donors were isolated from whole blood as describedabove.

The donors were three patients diagnosed with melanoma, three patientsdiagnosed with breast cancer, and three patients diagnosed withpancreatic cancer. Donor PBMC's were stimulated with a pool of peptidesof the common tumor antigen proteins (MAGEA1/MAGE3/NY-ESO) in thepresence or absence of increasing concentrations of SEA-CD40 or SGN-40for 5 days. Tissue culture supernatants were collected and INF-γproduction was assessed. Results are shown in FIG. 13 . Six out of thenine patients exhibited an antigen dependent INF-γ response that wassignificantly enhanced by SEA-CD40 treatment as compared to treatmentwith SGN-40. In the SEA-CD40 treated PBMC's, stimulation was observed atconcentrations as low as 10 μg/ml.

PBMC's from donors were isolated from whole blood as described above. Asabove, the donors were three patients diagnosed with melanoma, threepatients diagnosed with breast cancer, and three patients diagnosed withpancreatic cancer. Donor PBMC's were stimulated with a pool of peptidesof the common tumor antigen proteins (MAGEA1/MAGE3/NY-ESO) in thepresence or absence of increasing concentrations of SEA-CD40 and/or aconstant concentration of an anti-CTLA4 or anti-PD1 blocking antibody.Results are shown in FIG. 14 . While SEA-CD40 and antibodies against thecheckpoint blockade targets PD1 and CTLA4 stimulated an antigen specificT-cell response alone, a robust signal to the tumor antigen as measuredby INF-γ production was observed when SEA-CD40 was combined with eitheranti-CTLA4 antibodies or anti-PD1 antibodies.

Example 5: Mouse Models for Activity of Non-Fucosylated Anti-CD40Antibodies

Mouse models have been proven to be very useful in assessing efficacyand mechanisms of new cancer therapeutics. Study of SEA-CD40 in mousemodels of cancer has been difficult because SEA-CD40 does not recognizemurine CD40. Therefore, to assess the activity of the non-fucosylatedanti-CD40 antibodies a syngeneic murine tumor model was developed. Themurine functional equivalents of human IgG1 and human FcγRIII/CD16 aremurine IgG2a and FcγRIV, respectively, and binding of murine IgG2a tomurine FcγRIV mediates ADCC. See, e.g., Bruhns, Blood 119:5640-5649(2012) and Nimmeriahn et al., Immunity 23:41-51 (2005). The rat antibody1C 10 was used to generate a surrogate of SEA-CD40. See, e.g., Heath etal., Eur. J. Immunol. 24:1828-1834 (1994). Briefly, the VL and VH genefragments of a rat monoclonal antibody that recognizes murine CD40, the1C10 antibody were cloned in-frame 5′ to murine Ckappa and murine IgG2aCH1-CH2-CH3 fragments, respectively. Expression of the resulting genesin CHO cells generated a chimeric 1C10 antibody with rat VL and VHdomains and murine light and heavy chain domains of the IgG2a isotype(mIgG2a 1C10). mIgG2a 1C10 was expressed in the presence of2-fluorofucose in the CHO cell growth medium using the methods describedin Example 1, to generate a non-fucosylated form of mIgG2a 1C10 (mIgG2aSEA 1C10). Fucosylated mIgG2a 1C10 and mIgG2a SEA 1C10 were tested foranti-tumor activity using a mouse B16 melanoma model.

Assessment of non-fucosylated murine antibody binding to murine Fcγreceptors: CHO cells stably expressing murine FcγRI or FcγRIV wereincubated with increasing concentrations of fucosylated mIgG2a 1C10 ornon-fucosylated mIgG2a 1C10 (mIgG2a SEA-1C10). Samples were washed and asaturating amount of PE-anti-mouse IgG was added and incubated with thesamples on ice for thirty minutes. Samples were washed again and labeledcells were analyzed by flow cytometry.

Results: Binding of the surrogate anti-CD40 antibodies to Chinesehamster ovary (CHO) cells expressing the murine FcγRI or FcγRIV (themurine equivalent to human FcγRIII/CD16) was assessed. Results are showin FIGS. 15A and 15B. As expected mIgG2a 1C10 bound with similaraffinity as mIgG2a SEA 1C10 to FcγR1. See, e.g., FIG. 15A. However,non-fucosylated mIgG2a SEA 1C10 bound to FcγRIV at significantly higheraffinity than the fucosylated parental antibody mIgG2a 1C10. See, e.g.,FIG. 15B.

Assessment of non-fucosylated anti-CD40 antibodies in a murine tumormodel: 250.0E+3 B16F10 melanoma cells were given subcutaneously toC57BL/6 mice. Mice were randomized into cohorts each with tumor size ofapproximately 50 mm³ on average. Mice were then given interperitonealinjections of either an isotype control (mIgG2a), fucosylated mIgG2a1C10, or non-fucosylated mIgG2a 1C10 (mIgG2a SEA 1C10), every other dayfor a total of three doses. Mice were monitored until the tumor sizereached 1000 mm³, at which point the mice were sacrificed.

Results: The B16F10 sygeneic melanoma model was used to assess the invivo efficacy of our non-fucosylated anti-CD40 antibody surrogates.C57BL/6 mice were implanted with B16F10 melanoma cells, and then treatedwith either mIgG2a isotype control, fucosylated mIgG2a 1C10, ornon-fucosylated mIgG2a 1C10 (mIgG2a SEA 1C10). Tumor burden wasmonitored and mice were sacrificed when the tumor size reached 1000 mm³.Results are shown in FIG. 16 . Mice treated with non-fucosylatedSEA-1C10 mIgG2a showed a significant survival benefit and tumor delaycompared to the fucosylated parent 1C10 IgG2a antibody.

Example 6: SEA-CD40 Depletes B-Cells and Promotes T-Cell Activation

SEA-CD40 activity was compared to a related fucosylated antibody and toa fully agonistic anti-CD40 antibody, clone 21.4.1. Antibody 21.4.1 is ahuman anti-CD40 IgG2k agonistic antibody that is the parent clone ofCP-870,893, an antibody that is currently being tested in a clinicaltrial of solid tumors in combination with PDL1. For amino acid sequenceinformation for antibody 21.4.1, see, e.g., U.S. Pat. No. 7,338,660,which is herein incorporated for all purposes. Three functional areaswere tested for the antibodies: ability to drive human B-cellsdifferentiation, activation, and depletion, ability to activate primaryhuman PBMC cultures, and ability to drive an antigen specific response.

Assessment of B-cell activation by anti-CD-40 antibodies: Experimentswere performed using purified B-cells from fresh human whole blood orhuman peripheral blood mononuclear cells (PBMCs). B-cells were isolatedfrom fresh human whole blood using ROSETTESEP™ isolation kit. Theisolated, purified B-cells were cultured with increasing concentrationsof SEA-CD40, antibody 21.4.1, or hexameric CD40 ligand, ENZO LIFESCIENCES™ (10, 1, 0.1, 0.01, or 0.001 μg/mL) for 24 hours. B-cellactivation was assessed as upregulation of CD80 by Flow Cytometry.

PBMCs were isolated as described above and were then cultured withincreasing concentrations of SEA-CD40, antibody 21.4.1, or hexamericCD40L (10, 1, 0.1, 0.01, or 0.001 μg/mL) for 24 hours. The total numberof B-cells assessed with CD19 staining assessed by flow cytometry.

Results: SEA-CD40 immune modulatory activity is dependent on the Fcportion of the antibody and its interaction with the CD16, the FcγRIIIreceptor. Results are shown in FIG. 17. SEA-CD40 does not induce B-cellactivation in purified B-cells cultures which lack cells that expressthe Fcγ receptors needed for crosslinking of SEA-CD40. This differs fromthe CD40 activating antibody 21.4.1 which is able to drive B-cellactivation in pure B-cell cultures similar to CD40 ligand.

PBMCs include cells that express the Fcγ receptors. Results for thatcell population are shown in FIG. 18 . For PBMC cultures, SEA-CD40 wasable to promote ADCC depletion of B-cells, while antibody 21.4.1treatment did not deplete B-cells.

Assessment of monocyte/macrophage activation by anti-CD-40 antibodies:Human PBMC cultures were isolated as described above. PBMC cultures werestimulated with increasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0,or 10 μg/mL) of SEA-CD40, dacetuzumab, antibody 21.4.1, or anSEA-isotype control for twenty-four hours. Upregulation of CD80 is amarker of monocyte maturation. Surface expression of CD80 was assessedby flow cytometry.

Results: Results are shown in FIG. 19 . SEA-CD40 treatment of PBMCsinduces robust activation of monocyte/macrophages as measured by CD80up-regulation and this activity is on par with the activation seen withthe CD40 activating antibody 21.4.1.

Assessment of cytokine induction by anti-CD40 antibodies: Human PBMCcultures were isolated as described above. PBMC cultures were stimulatedwith increasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0, or 10 μg/mL)of SEA-CD40, dacetuzumab, antibody 21.4.1, or an SEA-isotype control fortwenty-four hours. Following stimulation, tissue culture supernatantswere collected and inflammatory cytokines assessed by multiplexedLUMINEX™ analysis.

Results: Results are shown in FIG. 20 and FIG. 21 . FIG. 20 shows thatSEA-CD40 and the CD40 activating antibody 21.4.1 induce cytokines IFN-γand chemokines important for eliciting robust T-cell responses. FIG. 21shows the induction of interleukin 10 (IL10) by the antibodies. Incontrast to antibody 21.4.1, which promotes IL10 production, SEA-CD40reduces the levels of the immune dampening cytokine IL-10.

Assessment of T-cell induction by anti-CD40 antibodies: Human PBMCcultures were isolated as described above. 1×10⁶ PBMCs were cultured inDMEM+10% FBS and incubated with 5 ug of M1 flu peptide, and withincreasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0, or 10 μg/mL) ofSEA-CD40, dacetuzumab, or antibody 21.4.1 for five days. Cells and cellculture supernatants were then collected. IFN-γ levels were assessed insupernatants. Flu antigen-specific T-cells assessed by tetramer stainingusing by flow cytometry. Percent T-regulatory cells, a CD4+, CD25+,CD127 low population of cells was determined using flow cytometry.

Results: Results are shown in FIGS. 22-24 . After the five dayincubation, SEA-CD40 induced higher levels of IFN-γ, as compared todacetuzumab or antibody 21.4.1, see, e.g., FIG. 22 . FIG. 23 shows thatSEA-CD40 induces a robust flu antigen specific T-cell response, similarto that seen with antibody 21.4.1. However, FIG. 24 shows that SEA-CD40also reduces the number of immune inhibitory T-regulatory cells presentafter flu peptide stimulation. This activity is likely related to thedecreased IL10 production seen after treatment of PBMCs with SEA-CD40.In contrast, after incubation with antibody 21.4.1, PBMCs showedincreased numbers of T-regulatory cells, as demonstrated in FIG. 24 .

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

INFORMAL SEQUENCE LISTING SEQ ID NO: 1; hS2C6 heavy chainEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSY        70        80        90       100       110       120    |    |    |    |    |    |    |    |    |    |    |    |NQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKGP       130       140       150       160       170       180    |    |    |    |    |    |    |    |    |    |    |    |SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS       190       200       210       220       230       240    |    |    |    |    |    |    |    |    |    |    |    |SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF       250       260       270       280       290       300    |     |    |     |    | †   |    |    |     |     |   *|PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV       310       320       330       340       350       360    |    |    |    |    |    |    |    |    |    |    |    |SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV       370       380       390       400       410       420    |    |    |    |    |    |    |    |    |    |    |    |SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF       430       440     |    |    |    | SCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 2, hS2C6 light chainDIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQKPGKAPKLLIYTVSNRF        70        80        90       100       110       120    |    |    |    |    |    |    |    |    |    |    |    |SGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSV       130       140       150       160       170       180    |    |    |    |    |    |    |    |    |    |    |    |FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL       190       200       210     |    |    |    |    |    |    |   SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

What is claimed is:
 1. A method of treating cancer, the methodcomprising the steps of administering an anti-CD40 antibody to a patientin need of such treatment, wherein the anti-CD40 antibody comprises theheavy chain variable region of SEQ ID NO:1 and the light chain variableregion of SEQ ID NO:2, and a human constant region; wherein the constantregion has an N-glycoside-linked sugar chain at residue N297 accordingto the EU index as set forth in Kabat and less than 5% of theN-glycoside-linked sugar chains comprise a fucose residue; and whereinthe anti-CD40 antibody is administered at a dose level between 0.1-2000μg/kg patient body weight.
 2. The method of claim 1, wherein the doselevel is between 10-1000 μg/kg.
 3. The method of claim 1, wherein thedose level is between 50-800 μg/kg.
 4. The method of claim 1, whereinthe dose level is between 75-600 μg/kg.
 5. The method of claim 1,wherein the dose level is between 100-500 μg/kg.
 6. The method of claim1, wherein the dose level is a range selected from the group consistingof 100-300 μg/kg, 300-500 μg/kg, 500-700 μg/kg, 700-900 μg/kg, and900-1100 μg/kg.
 7. The method of claim 1, wherein the dose level is arange selected from the group consisting of 100-150 μg/kg, 150-200μg/kg, 200-250 μg/kg, 250-300 μg/kg, 300-350 μg/kg, 350-400 μg/kg,400-450 μg/kg, 450-500 μg/kg, 500-550 μg/kg, 550-600 μg/kg, 600-650μg/kg, 650-700 μg/kg, 700-750 μg/kg, 750-800 μg/kg, 800-850 μg/kg,850-900 μg/kg, 900-950 μg/kg, 950-1000 μg/kg, 1000-1050 μg/kg, and1050-1100 μg/kg.
 8. The method of claim 1, wherein the dose level is amember of the group consisting of about 60 μg/kg, about 100 μg/kg, about150 μg/kg, about 200 μg/kg, about 250 μg/kg, about 300 μg/kg, about 350μg/kg, about 400 μg/kg, about 450 μg/kg, about 500 μg/kg, about 550μg/kg, about 600 μg/kg, about 650 μg/kg, about 700 μg/kg, about 750μg/kg, about 800 μg/kg, about 850 μg/kg, about 900 μg/kg, about 950μg/kg, about 1000-1050 μg/kg, about 1050 μg/kg, and 1110 μg/kg.
 9. Themethod of claim 1, wherein the anti-CD40 antibody is administered everythree weeks.
 10. The method of claim 1, wherein the anti-CD40 antibodyis administered every six weeks.
 11. The method of claim 1, wherein thepatient has a CD40 positive cancer.
 12. The method of claim 1, whereinthe patient has a CD40 negative cancer.
 13. A method of treating cancer,the method comprising the steps of administering an anti-CD40 antibodyand an anti-CTLA4 antibody to a patient in need of such treatment,wherein the anti-CD40 antibody comprises the heavy chain variable regionof SEQ ID NO:1 and the light chain variable region of SEQ ID NO:2, and ahuman constant region; wherein the constant region glycosylated atresidue N297 according to the EU index as set forth in Kabat and lessthan 5% of the glycosylated things comprise a fucose residue.
 14. Themethod of claim 13, wherein the anti-CTLA4 antibody is selected from thegroup consisting of ipilimumab and tremelimumab.
 15. A method oftreating cancer, the method comprising the steps of administering ananti-CD40 antibody and an anti-PD1 antibody to a patient in need of suchtreatment, wherein the anti-CD40 antibody comprises the heavy chainvariable region of SEQ ID NO:1 and the light chain variable region ofSEQ ID NO:2, and a human constant region; wherein the constant regionglycosylated at residue N297 according to the EU index as set forth inKabat and less than 5% of the glycosylated things comprise a fucoseresidue.
 16. The method of claim 15, wherein the anti-PD1 antibody isselected from the group consisting of nivolumab, pidilizumab, andpembrolizumab.
 17. A method of treating cancer, the method comprisingthe steps of administering an anti-CD40 antibody and an anti-PD-L1antibody to a patient in need of such treatment, wherein the anti-CD40antibody comprises the heavy chain variable region of SEQ ID NO:1 andthe light chain variable region of SEQ ID NO:2, and a human constantregion; wherein the constant region glycosylated at residue N297according to the EU index as set forth in Kabat and less than 5% of theglycosylated things comprise a fucose residue.
 18. The method of claim17, wherein the anti-PD-L1 antibody is selected from the groupconsisting of MEDI4736 and MPDL3280A.
 19. The method of claim 1, whereinthe cancer is a hematologic cancer.
 20. The method of claim 1, whereinthe cancer is a solid tumor.
 21. The method of claim 13, 15 or 17,wherein the cancer is a hematologic cancer.
 22. The method of claim 13,15 or 17, wherein the cancer is a solid tumor.